Global DNA hypomethylation suppresses squamous carcinogenesis in the tongue and esophagus

Abstract

Genome-wide DNA hypomethylation and concomitant site-specific gene hypermethylation are among the most common molecular alterations in human neoplasia. Previous studies revealed that genetic reduction of the DNA methylation level results in opposing effects on tumor development, depending on the tumor cell type and on the different stages of the tumorigenesis. For instance, reduced levels of DNA methylation in mice strongly inhibited tumor development of the intestine, whereas they induced thymic lymphomas and liver tumors. In the present study, using DNA methyltrasferase 1 (Dnmt1) hypomorphic alleles to reduce genomic methylation, we examined the effects of DNA hypomethylation on a murine squamous carcinogenesis in the tongue and esophagus induced by 4-nitroquinoline 1-oxide. Genetic reduction of DNA methylation level led to the suppression of tumor formation in both tongue and esophagus. Histological analyses revealed that DNA hypomethylation preferentially inhibited the development of squamous cell carcinomas. The results suggest that genomic hypomethylation inhibits squamous carcinogenesis in the tongue and esophagus, and that pharmacological modification of epigenetic status might be useful for the prevention and treatment of cancers in the upper digestive tract.

Figure 1

Representative macroscopic appearance of the tongue and histology of different stages of squamous lesions. (a–c) Macroscopic appearance of the tongue. (a) 4‐Nitroquinoline 1‐oxide (4NQO)‐exposed DNA methyltrasferase 1 (Dnmt1)^chip/+, (b) 4NQO‐exposed Dnmt1 ^chip/c, and (c) the control Dnmt1 ^chip/+ mouse. Nodular and polypoid tumors are seen in the dorsum and tip of the tongue. (d–f) A histological sequence of epithelial changes from dysplasia through invasive squamous cell carcinoma. (d) Dysplasia, (e) papilloma, and (f) squamous cell carcinoma observed in the present study. Scale bars = 100 µm.

Figure 2

Percentage of Ki‐67‐positive cells in non‐tumoral squamous epithelium of both DNA methyltrasferase 1 (Dnmt1)^chip/+ and Dnmt1 ^chip/c mice. (a) The Ki‐67‐positive ratio at the non‐tumoral squamous epithelium of the tongue. 4‐Nitroquinoline 1‐oxide (4NQO) administration resulted in an increased ratio of Ki‐67‐positive cells in squamous epithelium at the dorsum and tip of the tongue where direct exposure of 4NQO is expected. In contrast, the ratio at the inferior surface where 4NQO exposure is expected to be less than other sites did not change. The Ki‐67‐positive ratio is significantly lower in Dnmt1 ^chip/c mice in comparison with Dnmt1 ^chip/+ mice, suggesting that DNA hypomethylation suppresses cell proliferative activity. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. (b) The Ki‐67‐positive ratio at the non‐tumoral squamous epithelium of the esophagus. The Ki‐67‐positive ratio in Dnmt1 ^chip/c mice exposed to 4NQO is significantly lower than that in Dnmt1 ^chip/+ mice exposed to 4NQO. *P < 0.001.

Figure 3

Expansion of the CK19‐positive cell layer in 4‐nitroquinoline 1‐oxide (4NQO)‐exposed tongue epithelium and its suppression by genomic hypomethylation. (a–c) CK19 immunohistological staining of tongue squamous epithelium. (a) 4NQO non‐exposed DNA methyltrasferase 1 (Dnmt1) ^chip/+ mice. (b) 4NQO exposed Dnmt1 ^chip/+ mice. (c) 4NQO exposed Dnmt1^chip/c mice. Note that 4NQO exposure resulted in increased thickness of the CK19‐positive layer, whereas such an effect was less prominent in DNA hypomethylated mice. Scale bars = 100 µm. (d) Thickness of CK19‐positive cells (µm). (e) The CK19‐positive ratio at the dorsum of the tongue. *P < 0.001, **P < 0.005, ***P < 0.05.